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The article focuses on determining the function of nine genes implicated in COPD by GWAS in induced pluripotent stem cell–derived type 2 alveolar epithelial cells (iAT2s) by applying CRISPR. Topics discussed include biology of potential genetic contributors in the lung to COPD, including genome-wide association studies (GWAS) genes, and CRISPRi-mediated knockdown of COPD GWAS genes alters the iAT2 transcriptional program.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia. [ABSTRACT FROM AUTHOR]

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma. [ABSTRACT FROM AUTHOR]

Type-2 immunity elicits tissue repair and homeostasis, however dysregulated type-2 responses cause aberrant tissue remodelling, as observed in asthma. Severe respiratory viral infections in infancy predispose to later asthma, however, the processes that mediate tissue damage-induced type-2 inflammation and the origins of airway remodelling remain ill-defined. Here, using a preclinical mouse model of viral bronchiolitis, we find that increased epithelial and mesenchymal high-mobility group box 1 (HMGB1) expression is associated with increased numbers of IL-13-producing type-2 innate lymphoid cell (ILC2s) and the expansion of the airway smooth muscle (ASM) layer. Anti-HMGB1 ablated lung ILC2 numbers and ASM growth in vivo, and inhibited ILC2-mediated ASM cell proliferation in a co-culture model. Furthermore, we identified that HMGB1/RAGE (receptor for advanced glycation endproducts) signalling mediates an ILC2-intrinsic IL-13 auto-amplification loop. In summary, therapeutic targeting of the HMGB1/RAGE signalling axis may act as a novel asthma preventative by dampening ILC2-mediated type-2 inflammation and associated ASM remodelling. Author summary: Asthma can start at any time in life, although most often begins in early childhood. Wheezy viral bronchiolitis is a major independent risk factor for subsequent asthma. However, key knowledge gaps exist in relation to the sequelae of severe viral bronchiolitis and the pathogenic processes that promote type-2 inflammation and airway wall remodelling, cardinal features of asthma. Our study addresses this gap by identifying high-mobility group box 1 as a pathogenic cytokine that contributes to group 2 innate lymphoid cell-induced airway smooth muscle growth. [ABSTRACT FROM AUTHOR]

Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma. [ABSTRACT FROM AUTHOR]

RSV-induced prostaglandin D2 release contributes to disease severity by impairing IFN-λ production. RSV gives innate immunity the runaround: Asthma can be exacerbated by pathogens such as respiratory syncytial virus (RSV); prostaglandin D2 (PGD2) is also important in asthma and is being investigated as a therapeutic target. Werder et al. used multiple models to examine how RSV infection may perturb immune responses and influence asthma pathogenesis. Samples from infants with bronchiolitis or primary pediatric epithelial cells infected with RSV had elevated PGD2. Modulating PGD2 signaling in a mouse model of severe bronchiolitis improved antiviral immunity and dampened asthmatic symptoms later in life. This protection was not due to preventing type 2 immunity but instead a restoration of IFN-λ production. Their findings shed light on this host-pathogen interaction and suggest new therapeutic avenues. Prostaglandin D2 (PGD2) signals through PGD2 receptor 2 (DP2, also known as CRTH2) on type 2 effector cells to promote asthma pathogenesis; however, little is known about its role during respiratory syncytial virus (RSV) bronchiolitis, a major risk factor for asthma development. We show that RSV infection up-regulated hematopoietic prostaglandin D synthase expression and increased PGD2 release by cultured human primary airway epithelial cells (AECs). Moreover, PGD2 production was elevated in nasopharyngeal samples from young infants hospitalized with RSV bronchiolitis compared to healthy controls. In a neonatal mouse model of severe viral bronchiolitis, DP2 antagonism decreased viral load, immunopathology, and morbidity and ablated the predisposition for subsequent asthma onset in later life. This protective response was abolished upon dual DP1/DP2 antagonism and replicated with a specific DP1 agonist. Rather than mediating an effect via type 2 inflammation, the beneficial effects of DP2 blockade or DP1 agonism were associated with increased interferon-λ (IFN-λ) [interleukin-28A/B (IL-28A/B)] expression and were lost upon IL-28A neutralization. In RSV-infected AEC cultures, DP1 activation up-regulated IFN-λ production, which, in turn, increased IFN-stimulated gene expression, accelerating viral clearance. Our findings suggest that DP2 antagonists or DP1 agonists may be useful antivirals for the treatment of viral bronchiolitis and possibly as primary preventatives for asthma. [ABSTRACT FROM AUTHOR]

Severe viral lower respiratory infections are a major cause of infant morbidity. In developing countries, respiratory syncytial virus (RSV)-bronchiolitis induces significant mortality, whereas in developed nations the disease represents a major risk factor for subsequent asthma. Susceptibility to severe RSV-bronchiolitis is governed by gene-environmental interactions that affect the host response to RSV infection. Emerging evidence suggests that the excessive inflammatory response and ensuing immunopathology, typically as a consequence of insufficient immunoregulation, leads to long-term changes in immune cells and structural cells that render the host susceptible to subsequent environmental incursions. Thus, the initial host response to RSV may represent a tipping point in the balance between long-term respiratory health or chronic disease (e.g., asthma). The composition and diversity of the microbiota, which in humans stabilizes in the first year of life, critically affects the development and function of the immune system. Hence, perturbations to the maternal and/or infant microbiota are likely to have a profound impact on the host response to RSV and susceptibility to childhood asthma. Here, we review recent insights describing the effects of the microbiota on immune system homeostasis and respiratory disease and discuss the environmental factors that promote microbial dysbiosis in infancy. Ultimately, this knowledge will be harnessed for the prevention and treatment of severe viral bronchiolitis as a strategy to prevent the onset and development of asthma. [ABSTRACT FROM AUTHOR]

COPD is a major cause of morbidity and mortality globally. While the significance of environmental exposures in disease pathogenesis is well established, the functional contribution of genetic factors has only in recent years drawn attention. Notably, many genes associated with COPD risk are also linked with lung function. Because reduced lung function precedes COPD onset, this association is consistent with the possibility that derangements leading to COPD could arise during lung development. In this review, we summarise the role of leading genes ( HHIP , FAM13A , DSP , AGER and TGFB2 ) identified by genome-wide association studies in lung development and COPD. Because many COPD genome-wide association study genes are enriched in lung epithelial cells, we focus on the role of these genes in the lung epithelium in development, homeostasis and injury., Competing Interests: Conflict of interest: X. Zhou has received grant support from Bayer. M.H. Cho has received grant support from GSK and Bayer; and has received speaking/consulting fees from Illumina, AstraZeneca and Genentech. R.B. Werder and A.A. Wilson declared no conflicts of interest., (Copyright ©The authors 2024.)

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as "matrix"), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 10 30 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPC tdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

Type 2 alveolar epithelial cells (AT2s), facultative progenitor cells of the lung alveolus, play a vital role in the biology of the distal lung. In vitro model systems that incorporate human cells, recapitulate the biology of primary AT2s, and interface with the outside environment could serve as useful tools to elucidate functional characteristics of AT2s in homeostasis and disease. We and others recently adapted human induced pluripotent stem cell-derived AT2s (iAT2s) for air-liquid interface (ALI) culture. Here, we comprehensively characterize the effects of ALI culture on iAT2s and benchmark their transcriptional profile relative to both freshly sorted and cultured primary human fetal and adult AT2s. We find that iAT2s cultured at ALI maintain an AT2 phenotype while upregulating expression of transcripts associated with AT2 maturation. We then leverage this platform to assay the effects of exposure to clinically significant, inhaled toxicants including cigarette smoke and electronic cigarette vapor.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 ( ACE2 ), and transmembrane protease serine 2 ( TMPRSS2 ). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y 13 -R (P2Y 13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y 13 -R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y 13 -R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y 13 -R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y 13 -R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y 13 -R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y 13 -R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.

Prostaglandin D2 (PGD2) signals through PGD2 receptor 2 (DP2, also known as CRTH2) on type 2 effector cells to promote asthma pathogenesis; however, little is known about its role during respiratory syncytial virus (RSV) bronchiolitis, a major risk factor for asthma development. We show that RSV infection up-regulated hematopoietic prostaglandin D synthase expression and increased PGD2 release by cultured human primary airway epithelial cells (AECs). Moreover, PGD2 production was elevated in nasopharyngeal samples from young infants hospitalized with RSV bronchiolitis compared to healthy controls. In a neonatal mouse model of severe viral bronchiolitis, DP2 antagonism decreased viral load, immunopathology, and morbidity and ablated the predisposition for subsequent asthma onset in later life. This protective response was abolished upon dual DP1/DP2 antagonism and replicated with a specific DP1 agonist. Rather than mediating an effect via type 2 inflammation, the beneficial effects of DP2 blockade or DP1 agonism were associated with increased interferon-λ (IFN-λ) [interleukin-28A/B (IL-28A/B)] expression and were lost upon IL-28A neutralization. In RSV-infected AEC cultures, DP1 activation up-regulated IFN-λ production, which, in turn, increased IFN-stimulated gene expression, accelerating viral clearance. Our findings suggest that DP2 antagonists or DP1 agonists may be useful antivirals for the treatment of viral bronchiolitis and possibly as primary preventatives for asthma., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Respiratory syncytial virus-bronchiolitis is a major independent risk factor for subsequent asthma, but the causal mechanisms remain obscure. We identified that transient plasmacytoid dendritic cell (pDC) depletion during primary Pneumovirus infection alone predisposed to severe bronchiolitis in early life and subsequent asthma in later life after reinfection. pDC depletion ablated interferon production and increased viral load; however, the heightened immunopathology and susceptibility to subsequent asthma stemmed from a failure to expand functional neuropilin-1 + regulatory T (T reg) cells in the absence of pDC-derived semaphorin 4a (Sema4a). In adult mice, pDC depletion predisposed to severe bronchiolitis only after antibiotic treatment. Consistent with a protective role for the microbiome, treatment of pDC-depleted neonates with the microbial-derived metabolite propionate promoted Sema4a-dependent T reg cell expansion, ameliorating both diseases. In children with viral bronchiolitis, nasal propionate levels were decreased and correlated with an IL-6 high /IL-10 low microenvironment. We highlight a common but age-related Sema4a-mediated pathway by which pDCs and microbial colonization induce T reg cell expansion to protect against severe bronchiolitis and subsequent asthma., (© 2018 Lynch et al.)

Memory Th2 cell responses underlie the development and perpetuation of allergic diseases. Because these states result from immune dysregulation, established Th2 cell responses represent a significant challenge for conventional immunotherapies. New approaches that overcome the detrimental effects of immune dysregulation are required. We tested whether memory Th2 cell responses were silenced using a therapeutic approach where allergen expression in DCs is transferred to sensitized recipients using BM cells as a vector for therapeutic gene transfer. Development of allergen-specific Th2 responses and allergen-induced airway inflammation was blocked by expression of allergen in DCs. Adoptive transfer studies showed that Th2 responses were inactivated by a combination of deletion and induction of T cell unresponsiveness. Transfer of BM encoding allergen expression targeted to DCs terminated, in an allergen-specific manner, Th2 responses in sensitized recipients. Importantly, when preexisting airway inflammation was present, there was effective silencing of Th2 cell responses, airway inflammation was alleviated, and airway hyperreactivity was reversed. The effectiveness of DC-targeted allergen expression to terminate established Th2 responses in sensitized animals indicates that exploiting cell-intrinsic T cell tolerance pathways could lead to development of highly effective immunotherapies.

Respiratory syncytial virus (RSV)-bronchiolitis is a major cause of infant morbidity and mortality and a risk factor for subsequent asthma. We showed previously that toll-like receptor (TLR)7 in plasmacytoid dendritic cells (pDCs) is critical for protection against bronchiolitis and asthma in mice infected with pneumonia virus of mice (PVM), the mouse homolog of RSV. This lack of redundancy was unexpected as interferon-β promotor stimulator-1 (IPS-1) signalling, downstream of RIG-I-like receptor (RLR) and not TLR7 activation, contributes to host defence in hRSV-inoculated adult mice. To further clarify the role of IPS-1 signalling, we inoculated IPS-1 -/- and WT mice with PVM in early-life, and again in later-life, to model the association between bronchiolitis and asthma. IPS-1 deficiency predisposed to severe PVM bronchiolitis, characterised by neutrophilic inflammation and necroptotic airway epithelial cell death, high mobility group box 1 (HMGB1) and IL-33 release, and downstream type-2 inflammation. Secondary infection induced an eosinophilic asthma-like pathophysiology in IPS-1 -/- but not WT mice. Mechanistically, we identified that IPS-1 is necessary for pDC recruitment, IFN-α production and viral control. Our findings suggest that TLR7 and RLR signalling work collaboratively to optimally control the host response to pneumovirus infection thereby protecting against viral bronchiolitis and subsequent asthma.

Acute lower respiratory tract infections (ALRTI) are the leading cause of global childhood mortality, with human respiratory syncytial virus (hRSV) being a major cause of viral ALRTI in young children worldwide. In sub-Saharan Africa, many young children experience severe illnesses due to hRSV or Plasmodium infection. Although the incidence of malaria in this region has decreased in recent years, there remains a significant opportunity for coinfection. Recent data show that febrile young children infected with Plasmodium are often concurrently infected with respiratory viral pathogens but are less likely to suffer from pneumonia than are non-Plasmodium-infected children. Here, we hypothesized that blood-stage Plasmodium infection modulates pulmonary inflammatory responses to a viral pathogen but does not aid its control in the lung. To test this, we established a novel coinfection model in which mice were simultaneously infected with pneumovirus of mice (PVM) (to model hRSV) and blood-stage Plasmodium chabaudi chabaudi AS (PcAS) parasites. We found that PcAS infection was unaffected by coinfection with PVM. In contrast, PVM-associated weight loss, pulmonary cytokine responses, and immune cell recruitment to the airways were substantially reduced by coinfection with PcAS. Importantly, PcAS coinfection facilitated greater viral dissemination throughout the lung. Although Plasmodium coinfection induced low levels of systemic interleukin-10 (IL-10), this regulatory cytokine played no role in the modulation of lung inflammation or viral dissemination. Instead, we found that Plasmodium coinfection drove an early systemic beta interferon (IFN-β) response. Therefore, we propose that blood-stage Plasmodium coinfection may exacerbate viral dissemination and impair inflammation in the lung by dysregulating type I IFN-dependent responses to respiratory viruses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)